The salamander has the greatest regenerative ability of any limbed vertebrate, as salamanders can regenerate entire limbs, organs, and central nervous system structures after amputation or injury. The study of these remarkable feats of regeneration may guide human regenerative medicine, and the tools for this study are rapidly improving. Only recently have high-throughput sequencing techniques allowed scientists to begin to identify large numbers of genes that are highly enriched in regenerating tissues. Genome editing techniques now make it possible to investigate the function of these enriched genes in the salamander. I find that the CRISPR/Cas system of targeted mutagenesis can be easily applied to the Ambystoma mexicanum salamander, or the axolotl, to create individual animals with targeted loss-of-function of specific genes. I propose to use a novel approach to assess the contribution of specific genes to limb regeneration in the axolotl. I find that I can us a genome editing approach to simultaneously label individual populations of cells and alter the function of targeted genes. These two features of gene editing can accelerate the characterization of genes during limb regeneration in the axolotl. By combining gene editing with high-throughput sequencing, I propose to address three topics. I will determine whether a small set of cells give rise to all the structures of a regenerated limb, whether limb regeneration requires existing stores of stem cells, and whether uncharacterized genes are required for regeneration. This project may speed the translation of basic research into the biology of an exotic animal into therapeutics to improve healing in humans.